Large CRISPR-Cas-induced deletions in the oxamniquine resistance locus of the human parasiteSchistosoma mansoni

Abstract

At least 250 million people worldwide suffer from schistosomiasis, caused by Schistosoma worms. Genome sequences for several Schistosoma species are available, including a highquality annotated reference for Schistosoma mansoni. There is a pressing need to develop a reliable functional toolkit to translate these data into new biological insights and targets for intervention. CRISPR-Cas9 was recently demonstrated for the first time in S. mansoni, to produce somatic mutations in the omega-1 (ω1) gene. Here, we employed CRISPR-Cas9 to introduce somatic mutations in a second gene, SULT-OR, a sulfotransferase expressed in the parasitic stages of S. mansoni, in which mutations confer resistance to the drug oxamniquine. A 262-bp PCR product spanning the region targeted by the gRNA against SULT-OR was amplified, and mutations identified in it by high-throughput sequencing. We found that 0.3-2.0% of aligned reads from CRISPR-Cas9-treated adult worms showed deletions spanning the predicted Cas9 cut site, compared to 0.1-0.2% for sporocysts, while deletions were extremely rare in eggs. The most common deletion observed in adults and sporocysts was a 34 bp-deletion directly upstream of the predicted cut site, but rarer deletions reaching as far as 102 bp upstream of the cut site were also detected. The CRISPR-Cas9-induced deletions, if homozygous, are predicted to cause resistance to oxamniquine by producing frameshifts, ablating SULT-OR transcription, or leading to mRNA degradation via the nonsense-mediated mRNA decay pathway. However, no SULT-OR knock down at the mRNA level was observed, presumably because the cells in which CRISPR-Cas9 did induce mutations represented a small fraction of all cells expressing SULT-OR. Further optimisation of CRISPR-Cas protocols for different developmental stages and particular cell types, including germline cells, will contribute to the generation of a homozygous knock-out in any gene of interest, and in particular the SULT-OR gene to derive an oxamniquine-resistant stable transgenic line.

CRISPR/Cas9-mediated genome editing ofSchistosoma mansoniacetylcholinesterase

“…This suggests the NHEJ substitutions detected by CRISPResso2 are likely false positives introduced through PCR or sequencing errors although further study is required to confirm these observations. Deletions and insertions were rare in AChE-edited eggs, similar to the report by Sankaranarayanan et al [16], where CRISPR/Cas9 was used to edit the SULT-OR in S. mansoni eggs. A possible explanation for these results might be low expression levels of some key NHEJ repair enzymes required for CRISPR/Cas9 editing in schistosome eggs [16], including gene Smp_211060 identified in S. mansoni as a homolog of the KU70/KU80 genes which are essential for the NHEJ pathway [39][40][41].…”

“…Deletions and insertions were rare in AChE-edited eggs, similar to the report by Sankaranarayanan et al [16], where CRISPR/Cas9 was used to edit the SULT-OR in S. mansoni eggs. A possible explanation for these results might be low expression levels of some key NHEJ repair enzymes required for CRISPR/Cas9 editing in schistosome eggs [16], including gene Smp_211060 identified in S. mansoni as a homolog of the KU70/KU80 genes which are essential for the NHEJ pathway [39][40][41]. The limited NHEJ indels detected in AChE-edited eggs suggested the possibility that most DSBs in AChE loci can be repaired by re-ligating directly without insertion or deletions.…”

“…In this study, our NGS analysis focused on AChE-edited eggs. However, as reported by Sankaranarayanan et al [16], adult S. mansoni might be a better stage to achieve higher efficiency of CRISPR/Cas9 mediated modifications compared with eggs (and sporocysts). Accordingly, an improved programmed genome editing may occur in AChE-edited adults, which we now plan to investigate.…”

“…Incomplete modification of the AChE gene may stimulate S. mansoni to alter cholinergic signalling transduction and further regulate downstream signalling pathways (such as the PI3K and ERK pathways), thereby amplifying the effect of the knockdown. In addition, as reported in CRISPR/Cas9 editing in S. mansoni [16], Strongyloides [53] and C. elegans [54], large deletions might be missed by amplicon sequencing. Undetected modification(s) during AChE editing may have led to the pronounced phenotypic changes observed at the protein level in these helminths but this needs to be further explored.…”

“…Ittiprasert et al pioneered the CRISPR/Cas9 editing system in S. mansoni eggs, targeting a single gRNA of omega-1 (ω1), which plays a crucial role in Th2 polarization and granuloma formation [15]. Sankaranarayanan et al compared CRISPR/Cas9 mediated deletions in different life cycle stages of S. mansoni including adult worms, sporocysts and eggs, targeting the gene SULT-OR which is involved in oxamniquine resistance [16] (preprint data). Arunsan et al also undertook a similar approach to modify the granulin (Ov-GRN-1) gene in the liver fluke Opisthorchis viverrini which plays a key role in virulence morbidity during opisthorchiasis [17].…”